scholarly journals Campylobacter jejuni Serine Protease HtrA Cleaves the Tight Junction Component Claudin-8

2020 ◽  
Vol 10 ◽  
Author(s):  
Irshad Sharafutdinov ◽  
Delara Soltan Esmaeili ◽  
Aileen Harrer ◽  
Nicole Tegtmeyer ◽  
Heinrich Sticht ◽  
...  
Keyword(s):  
Tight Junction ◽  
Serine Protease ◽  
Campylobacter Jejuni ◽  
Adherens Junctions ◽  
Present Report ◽  
Acute Infection ◽  
Cell Junctions ◽  
Junction Protein ◽  
Carboxy Terminal

Campylobacter jejuni express the high temperature requirement protein A (HtrA), a secreted serine protease, which is implicated in virulence properties of the pathogen. Previous studies have shown that C. jejuni HtrA can cleave the epithelial transmembrane proteins occludin and E-cadherin in the tight and adherens junctions, respectively. In the present report, we studied the interaction of HtrA with another human tight junction protein, claudin-8. Confocal immunofluorescence experiments have shown that C. jejuni infection of the intestinal polarized epithelial cells in vitro leads to a relocation of claudin-8. Wild-type C. jejuni induced the downregulation of claudin-8 signals in the tight junctions and an accumulation of claudin-8 agglomerates in the cytoplasm, which were not seen during infection with isogenic ΔhtrA knockout deletion or protease-inactive S197A point mutants. Western blotting of protein samples from infected vs. uninfected cells revealed that an 18-kDa carboxy-terminal fragment is cleaved-off from the 26-kDa full-length claudin-8 protein, but not during infection with the isogenic ΔhtrA mutant. These results were confirmed by in vitro cleavage assays using the purified recombinant C. jejuni HtrA and human claudin-8 proteins. Recombinant HtrA cleaved purified claudin-8 in vitro giving rise to the same 18-kDa sized carboxy-terminal cleavage product. Mapping studies revealed that HtrA cleavage occurs in the first extracellular loop of claudin-8. Three-dimensional modeling of the claudin-8 structure identified an exposed HtrA cleavage site between the amino acids alanine 58 and asparagine 59, which is in well agreement with the mapping studies. Taken together, HtrA operates as a secreted virulence factor targeting multiple proteins both in the tight and adherens junctions. This strategy may help the bacteria to open the cell-to-cell junctions, and to transmigrate across the intestinal epithelium by a paracellular mechanism and establish an acute infection.

scholarly journals Paracingulin Regulates the Activity of Rac1 and RhoA GTPases by Recruiting Tiam1 and GEF-H1 to Epithelial Junctions

2008 ◽  
Vol 19 (10) ◽  
pp. 4442-4453 ◽  
Author(s):  
Laurent Guillemot ◽  
Serge Paschoud ◽  
Lionel Jond ◽  
Andrea Foglia ◽  
Sandra Citi
Keyword(s):  
Tight Junction ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Dependent Manner ◽  
Junction Protein ◽  
Junction Formation ◽  
Epithelial Junctions ◽  
Junctional Protein

Small GTPases control key cellular events, including formation of cell–cell junctions and gene expression, and are regulated by activating and inhibiting factors. Here, we characterize the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2. During tight junction assembly by the calcium-switch, Rac1 shows two transient peaks of activity, at earlier (10–20 min) and later (3–8 h) time points. Paracingulin depletion reduces such peaks of Rac1 activation in a Tiam1-dependent manner, resulting in a delay in junction formation. Paracingulin physically interacts with GEF-H1 and Tiam1 in vivo and in vitro, and it is required for their efficient recruitment to junctions, based on immunofluorescence and biochemical experiments. Our results provide the first description of a junctional protein that interacts with GEFs for both Rac1 and RhoA, and identify a novel molecular mechanism whereby Rac1 is activated during junction formation.

scholarly journals ZO-1 controls endothelial adherens junctions, cell–cell tension, angiogenesis, and barrier formation

2015 ◽  
Vol 208 (6) ◽  
pp. 821-838 ◽  
Author(s):  
Olga Tornavaca ◽  
Minghao Chia ◽  
Neil Dufton ◽  
Lourdes Osuna Almagro ◽  
Daniel E. Conway ◽  
...  
Keyword(s):  
Tight Junction ◽  
Adherens Junctions ◽  
Intercellular Junctions ◽  
Barrier Formation ◽  
Junction Protein ◽  
Endothelial Junctions ◽  
Tight Junction Disruption ◽  
Cell Cell

Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell–cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin–based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin–VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin–dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell–cell tension, migration, angiogenesis, and barrier formation.

scholarly journals ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

1998 ◽  
Vol 141 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Julie Haskins ◽  
Lijie Gu ◽  
Erika S. Wittchen ◽  
Jennifer Hibbard ◽  
Bruce R. Stevenson
Keyword(s):  
Amino Acid ◽  
Tight Junction ◽  
Molecular Mass ◽  
Mdck Cells ◽  
Cdna Libraries ◽  
Pdz Domains ◽  
Large Tumor ◽  
Junction Protein ◽  
Discs Large

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

scholarly journals Tight and Adherens Junctions in the Ovine Uterus: Differential Regulation by Pregnancy and Progesterone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3922-3931 ◽  
Author(s):  
M. Carey Satterfield ◽  
Kathrin A. Dunlap ◽  
Kanako Hayashi ◽  
Robert C. Burghardt ◽  
Thomas E. Spencer ◽  
...  
Keyword(s):  
Tight Junction ◽  
Adherens Junctions ◽  
Luminal Epithelium ◽  
Tissue Fluid ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Conceptus Development ◽  
Associated Proteins ◽  
Continuous Presence ◽  
D 12

In species with noninvasive implantation by conceptus trophectoderm, fetal/maternal communications occur across the endometrial epithelia. The present studies identified changes in junctional complexes in the ovine endometrium that regulate paracellular trafficking of water, ions, and other molecules, and the secretory capacity of the uterine epithelia. Distinct temporal and spatial alterations in occludin, tight junction protein 2, and claudin 1–4 proteins were observed in the endometrium of cyclic and early pregnant ewes. Dynamic changes in tight junction formation were characterized by an abundance of tight junction proteins on d 10 of the estrous cycle and pregnancy that substantially decreased by d 12. Early progesterone administration advanced conceptus development on d 9 and 12 that was associated with loss of tight-junction-associated proteins. Pregnancy increased tight-junction-associated proteins between d 14–16. Cadherin 1 and β-catenin, which form adherens junctions, were abundant in the endometrial glands, but decreased after d 10 of pregnancy in the luminal epithelium and then increased by d 16 with the onset of implantation. Results support the ideas that progesterone elicits transient decreases in tight and adherens junctions in the endometrial luminal epithelium between d 10–12 that increases selective serum and tissue fluid transudation to enhance blastocyst elongation, which is subsequently followed by an increase in tight and adherens junctions between d 14–16 that may be required for attachment and adherence of the trophectoderm for implantation. The continuous presence of tight and adherens junctions in the uterine glands would allow for vectorial secretion of trophic substances required for conceptus elongation and survival.

Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2451-2458 ◽  
Author(s):  
Amanda MacCallum ◽  
Simon P. Hardy ◽  
Paul H. Everest
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Campylobacter Jejuni ◽  
Fluid Transport ◽  
Cell Function ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ussing Chambers ◽  
Cell Monolayers ◽  
Junction Protein

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in ‘domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting (‘doming’) Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.

scholarly journals Layilin is critical for mediating hyaluronan 35 kDa-induced intestinal epithelial tight junction protein ZO-1 in vitro and in vivo

2018 ◽  
Vol 66 ◽  
pp. 93-109 ◽  
Author(s):  
Yeojung Kim ◽  
Gail A. West ◽  
Greeshma Ray ◽  
Sean P. Kessler ◽  
Aaron C. Petrey ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junction Protein ◽  
Intestinal Epithelial ◽  
Junction Protein ◽  
Epithelial Tight Junction

scholarly journals The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB

2006 ◽  
Vol 17 (3) ◽  
pp. 1322-1330 ◽  
Author(s):  
Anna Tsapara ◽  
Karl Matter ◽  
Maria S. Balda
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Tight Junction ◽  
Intracellular Signaling ◽  
Transcriptional Activity ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Nucleic Acid Binding ◽  
Junction Protein

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress.

WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment

2005 ◽  
Vol 289 (2) ◽  
pp. F431-F441 ◽  
Author(s):  
Maribel Rico ◽  
Amitava Mukherjee ◽  
Martha Konieczkowski ◽  
Leslie A. Bruggeman ◽  
R. Tyler Miller ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Slit Diaphragm ◽  
Cell Junctions ◽  
Puromycin Aminonucleoside ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Interacting Protein ◽  
Filtration Barrier ◽  
Cell Cell

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 μg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

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